Peptide having cell membrane penetrating activity

ABSTRACT

Provided are transmembrane complexes that contain a protein transduction domain (PTD) from the N-terminus of IgE-dependent histamine-releasing factor (HRF) and a target substance that is to be delivered into a cell. Also provided are nucleic acid molecules encoding the transmembrane complex, and methods of delivering the target substance into a cell interior by contacting the transmembrane complex with a cell. Also provided are transfection kits containing the PTD and the target substance.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/141,731, filed Apr. 28, 2016, which is a divisional of U.S.application Ser. No. 13/669,414, filed Nov. 5, 2012, which is acontinuation of U.S. application Ser. No. 12/280,077, filed Nov. 3,2008, which is the U.S. National Stage application of PCT/KR2007/000885,filed Feb. 20, 2007, which claims priority to Korean Patent ApplicationNo. 10-2006-0016156, filed Feb. 20, 2006, to Kyunglim Lee, Moonhee Kim,Miyoung Kim and Youngjoo Kwon. The subject matter of each of theabove-mentioned applications is incorporated by reference in itsentirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY

An electronic version of the Sequence Listing is filed herewith, thecontents of which are incorporated by reference in their entirety. Theelectronic file was created on Jan. 10, 2018, is 23 kilobytes in size,and titled 380ESEQ.US1.txt.

TECHNICAL FIELD

The present invention relates to a peptide having cell membranepenetrating activity, a transmembrane carrier comprising the peptidehaving cell membrane penetrating activity as an effective component, atransmembrane complex consisting of the peptide having cell membranepenetrating activity combined with a target substance, a transfectionkit comprising the peptide having cell membrane penetrating activity andthe target substance, use of the peptide having cell membranepenetrating activity for the manufacture of a transmembrane complex, useof the transmembrane complex for the manufacture of a medicament, and amethod for delivering a target substance into the cell interior whichcomprises administering to a subject a transmembrane complex consistingof the peptide having cell membrane penetrating activity combined with atarget substance to induce transduction of the transmembrane complexinto the cell interior.

BACKGROUND ART

Recently, various methods have been developed for deliveringmacromolecules such as therapeutic drug, peptides and proteins intocells in vitro and in vivo.

In vitro methods include electroporation, membrane fusion withliposomes, high velocity bombardment with DNA-coated microprojectiles,incubation with calcium-phosphate-DNA precipitate, DEAE-dextran mediatedtransfection, infection with modified viral nucleic acids, and directmicro-injection into single cells. But such methods are of extremelylimited usefulness for delivery of proteins.

Delivery of macromolecules into cells in vivo has been accomplished withscrape loading, calcium phosphate precipitates and liposomes. However,these techniques have, up to date, shown limited usefulness for in vivocellular delivery.

General methods for efficient delivery of biologically active proteinsinto intact cells, in vitro and in vivo include chemical addition of alipopeptide (P. Hoffmann et al., 1988) or a basic polymer such aspolylysine or polyarginine etc. (W-C. Chen et al., 1978)

Folic acid has been used as a transport moiety (C. P. Leamon and Low,1991). However, these methods have not proved to be highly reliable orgenerally useful.

Recently to introduce macromolecules such as a protein into a cellinterior, gene therapy becomes in the limelight but this have alsoproblems in that targeting is incorrect. As a alternative, research onprotein transduction or protein therapy is actively progressed.

Protein transduction domain (PTD) was first reported that purified humanimmunodeficiency virus type-1 (“HIV”) TAT protein is taken up from thesurrounding medium by adding it to human cells growing in culture medium(Green et al., 1988, Frankel et al., 1988). After this report,drosophila homeotic transcription factor, antennapedia (Antp) (Joliot etal., 1991) and herpes simplex virus-1 DNA-binding protein, VP22 (Elliotet al, 1997) were also identified.

In comparison of amino acid sequences of the PTDs such as TAT, Antp andVP22 etc., basic amino acids such as arginine and lysine exist for themost part (TABLE 1) and this sequence potentiates easy approach near tothe negatively charged phospholipid bilayer and penetration into thecell interior. Protein sequences having penetrating activity were namedas protein transduction domains (PTDs).

TABLE 1 PTD Amino Acid Sequences SEQ ID NO: HW-1 TATTyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg 82 HSV VP22Asp-Ala-Ala-Thr-Ala-Thr-Arg-Gly-Arg-Ser-Ala-Ala-Ser- 83Arg-Pro-Thr-Glu-Arg-Pro-Arg-Ala-Pro-Ala-Arg-Ser-Ala-Ser-Arg-Pro-Arg-Arg-Pro-Val-Glu AntpArg-Gln-Iso-Lys-Iso-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys- 84 Trp-Lys-Lys

In particular, recombinant expression vector was developed by using apeptide containing 11 amino acids of TAT 47-57 and TAT fusion proteinswere prepared by linking the TAT peptide to other peptides or proteinsand so introduction of full-length protein into intracellularcompartment became possible without the limitation of size or function(Nagahara et al., 1988).

As PTDs can be linked with other peptide or proteins to form fusionprotein and then be transduced into cell interior, there are manyattempts to transduce therapeutic drug, peptide, protein etc. into cellinterior using PTDs.

Recently, it has been known for PTDs which do not contain lots of basicamino acid residues. Also, it has been reported that PTDs penetratephospholipid bilayer of cell membrane by helix conformation.

TCTP (translationally controlled tumor protein) is a protein known asIgE-dependent histamine-releasing factor (HRF) as reported by MacDonaldet al. (1995). TCTP had been known as tumor-specific protein until 1980′and the synthesis thereof was assumed to be related to proliferativestage of tumor. TCTP was reported as a tumor protein of 21 kDa, p21 inmouse erythroleukemia cell line (Chitpatima et al., 1988). Also, it wasrevealed that p23, relating to cell growth in Ehrlich ascites tumor isthe same as TCTP/HRF (Bohm et al, 1989).

TCTP is frequently found in tumor cell, particularly growing vigorously,and exists in cytoplasm. It is a known protein consisting of 172 aminoacids (NCBI accession #P13693 (Homo sapiens)) and shows high homologybetween species. 45 amino acids at its C-terminal form basic domain.Because such domain has about 46% homology with MAP-1B,microtubule-associated protein, it was also assumed that HRF is amicrotubule-associated protein. Gachet, et al. (1997) observed that HRFis distributed consistently along with the cytoskeletal network to someextent using confocal microscope, which suggests that HRF binds to thecytoskeleton.

TCTP expression is characterized by that mRNA is maintained in regularlevel, but in case that exterior stimulus such as serum exists, it istransformed to polysome to be translated. According to thecharacteristic, it was named as ‘Translationally Controlled TumorProtein (TCTP)’(Thomas et al., 1981; Thomas and Thomas., 1986). It wasalso reported that TCTP mRNA is suppressed during translation, but whenit receives cell division signal, it is activated and translated toprotein (Thomas and Thomas, 1986).

TCTP/HRF is considered as a histamine releasing material interactingwith basophil or mast cell and related to allergic inflammatoryresponse.

MacDonald, et al. (1995) also found that though HRF is an intracellularprotein, HRF in the outside of cells stimulates IgE-sensitized basophilsto release histamine (Schroeder, et al., 1996). Schroeder, et al. (1997)observed that HRF can augment the anti-IgE-induced histamine releasefrom all basophils, regardless of the IgE absence, and thus suggestedthat HRF exerts its function by binding to cell membrane receptors, notby binding with IgE.

The present inventors have previously reported that TCTP/HRF isinteracted with third cytoplasmic domain(CD3) of subunit of (Na,K)ATPasethereby suppressing the activity of (Na,K)ATPase (as shown in KR PatentApplication No. 10-2001-0027896) (Jung et al., 2004).

At the same time the present inventors reports that TCTP/HRF can passthrough cell membrane. Since the amino acid sequence of TCTP/HRF has nothe part consisting of plenty of basic amino acids, arginine or lysine,which is a characteristic of representative PTDs, and no similar aminoacid sequences to those of other PTDs, the present inventors consideredTCTP has a domain which is different to other known PTDs in aspect ofthe protein structures.

In whole structure of TCTP, N- and C-terminus get loose and exposed andmiddle part forms a spherical shape.

In prediction of third structure, there are three helixes, wherein firsthelix(H1) is very short, second (H2) and third helix (H3) are exposed tooutside. By H2 and H3 structure of TCTP in Schizosaccharomyces pombe,basic amino acids are distributed to outside of helix (Thaw et al.,2001) and so H2 and H3 were predicted to be related to proteintransduction activity. However, by a test result, this helix part hadnothing to do with translocation.

Therefore if we identify amino acid sequences with protein transductionfunction in TCTP/HRF, it may be possible to find new types of PTD, aswell as to make a new drug delivery system though a novel vectordevelopment using these.

The present inventors made a constant effort for looking for PTD in TCTPand, as a result, isolated protein transduction domain composed of verydifferent amino acids in comparison with well-known PTDs. On the basisof this result, the present inventors have established the presentinvention by confirming that this domain shows remarkably high cellpenetrating activity than well-known PTDs.

DISCLOSURE OF INVENTION Technical Problem

It is an object of the present invention to provide a peptide havingcell membrane penetrating activity, a transmembrane carrier comprisingthe peptide having cell membrane penetrating activity as an effectivecomponent, a transmembrane complex consisting of the peptide having cellmembrane penetrating activity combined with a target substance, atransfection kit comprising the peptide having cell membrane penetratingactivity and the target substance, use of the peptide having cellmembrane penetrating activity for the manufacture of a transmembranecomplex, use of the transmembrane complex for the manufacture of amedicament, and a method for delivering a target substance into the cellinterior which comprises administering to a subject a transmembranecomplex consisting of the peptide having cell membrane penetratingactivity combined with a target substance to induce transduction of thetransmembrane complex into the cell interior.

Technical Solution

This invention provides a peptide having cell membrane penetratingactivity, composed of the following amino acid sequence:

R1-R2-R3-R4-R5-R6-R7-R8-R9-R10

In the above formula,

-   R1 may be deleted or one amino acid selected from M, A, Q, C, F, L    or W,-   R2 may be deleted or one amino acid selected from I or A,-   R3 may be one amino acid selected from I or A,-   R4 may be one amino acid selected from Y, A, F, S or R,-   R5 may be one amino acid selected from R, A or K,-   R6 may be one amino acid selected from D, A, I or R,-   R7 may be deleted or one amino acid selected from L, K, A, E or R,-   R8 may be deleted or one amino acid selected from I, K or A,-   R9 may be deleted or one amino acid selected from A, S, E, Y or T,-   R10 may be deleted or one amino acid selected from A, H, K or E, and-   if R10 is K or H, the amino acid(s) selected from K, KK, R, RR or HH    may be added thereto.

In one embodiment of the present invention, the amino acid sequence maybe SEQ ID NO:1.

In one embodiment of the present invention, the amino acid sequence maybe SEQ ID NOS:2-7.

Also, in one embodiment of the present invention, the amino acidsequence may be an amino acid sequence which one amino acid of SEQ IDNO:2 is substituted with alanine. The above amino acid sequence may be,for example, an amino acid sequence selected from SEQ ID NOS:8-16,particularly SEQ ID NO:13.

In addition, in an embodiment of the present invention, the amino acidsequence may be an amino acid sequence selected from SEQ ID NOS:20-54.The above sequence may be, for example, an amino acid sequence selectedfrom SEQ ID NOS:22, 26, 27 or SEQ ID NOS:31-54.

In the present invention, ‘cell membrane penetrating protein domain’means protein sequence having penetrating activity into cell interior(cytoplasm, nucleus) across plasma membrane.

A peptide having cell membrane penetrating activity of the presentinvention is a novel cell membrane penetrating protein domain that hasno similarity in sequences with well-known TAT, VP22 and AntpPTDs(Protein Transduction Domains).

The present invention provides a peptide having cell membranepenetrating activity consisting of the amino acid sequence of SEQ IDNO:1. The present invention also provides a peptide having cell membranepenetrating activity consisting of one amino acid sequence selected fromSEQ ID NOS:2-7.

According to one example of the present invention, the peptide havingcell membrane penetrating activity consisting of the amino acid sequenceof SEQ ID NOS:1, 2, 3 or 4 shows excellent cell penetrating activity incomparison with conventional TAT, and intracellular penetratingefficiency shows a rapidly increasing mode when treatment concentrationbecomes high and incubation time becomes long.

In detail, when cell penetrating activity was measured by using theresidues of TCTP from 1^(st) to 10^(th) [TCTP(1-10), SEQ ID NO:1], cellpenetrating activity of TCTP(1-10) show over 3 times activity whentreated for 15 minutes in 50 μM and 6 times activity when treated for 15minutes in 100 μM, compared to that of TAT. In case of treatment for 2hours, cell penetrating activity at concentration of 50 μM and 100 μM ofTCTP(1-10) were higher than those of TAT about 29 times and 30 times,respectively.

Also, compared with the case of treatment for 15 minutes, cellpenetrating activity showed an increased fashion in the incubation timeof 2 hours.

In addition, a peptide comprising amino acid residues of TCTP(1-9)(SEQID NO:2), TCTP(1-8)(SEQ ID NO:3) or TCTP(2-10)(SEQ ID NO:4) showed moreexcellent penetrating activity than well-known TAT(47-58) peptide. Ofthese, cell penetrating activity was excellent in the order ofTCTP(1-10)(SEQ ID NO:1), TCTP(1-9)(SEQ ID NO:2), TCTP(1-8)(SEQ ID NO:3)and TCTP(2-10)(SEQ ID NO:4), and when 1st amino acid of TCTP wasexisting, cell penetrating activity was more excellent.

Length of the peptides, as a common length of cell membrane penetratingprotein domain accepted in this art, may vary within the scope of,preferably, 9-15 residues, and more preferably, 9-10 residues.

A peptide having cell membrane penetrating activity of the presentinvention may be prepared by artificial synthesis or by isolating thesequence of TCTP(1-10)(SEQ ID NO:1), TCTP(1-9)(SEQ ID NO:2),TCTP(1-8)(SEQ ID NO:3) or TCTP(2-10)(SEQ ID NO:4) from TCTP.

Synthesis of the peptide of the present invention may be performed, forexample, by using an instrument or by using genetic engineering.

In case of synthesis by using an instrument, synthesis can be performedby using Fmoc solid-phase method on automatic peptide synthesizer(PeptrEX-R48, Peptron). After purifying the synthesized peptide fromresin, the peptide can be purified and analyzed by reverse-phase HPLC(Prominence LC-20AB, Shimadzu, Japan) with Shiseido capcell pak C18analytic RP column. After synthesis is completed, the peptide can beidentified by a mass spectrometer (HP 1100 Series LC/MSD,Hewlett-Packard, Roseville, USA).

In case of isolation by genetic engineering, nucleic acid sequencescorresponding to a desired peptide can be introduced into recombinantvector for protein expression, then the expression of peptide codingregion can be induced by IPTG in E. coli bacteria like a BL21(λDE3) orBL21(λDE3)pLys, that is deficient in proteases, and the peptide can bepurified.

The present invention also provides a peptide having cell membranepenetrating activity, composed of the amino acid sequence of SEQ IDNOS:8-16.

According to an example of the present invention, among the amino acidsequences that one amino acid of SEQ ID NO:2 is substituted withalanine, alanine-substituent of 6th residue, aspartic acid (SEQ IDNO:13), showed 2.5 times increased penetrating activity than WT(wildtype) peptide at a low concentration of 10 μM and alanine-substituentsof 5^(th) and 7-9^(th) residue (R, L, I, S)(SEQ ID NOS:12, 14-16) showeda little decreased but still showed activity. Activity ofalanine-substituents of 1^(st)-4^(th) residues (M, I, I, Y)(SEQ IDNOS:8-11) was suddenly decreased but maintained functionally like a WTpeptide. Therefore, a peptide having cell membrane penetrating activityof the present invention comprises the peptide consisting of one aminoacid sequence selected from SEQ ID NOS:8-16.

The present invention also provides a peptides having cell membranepenetrating activity, consisting of one amino acid sequence selectedfrom SEQ ID NOS:22, 26, 27, or 31-54.

In an example of the present invention, the peptides of SEQ ID NOS:20-30were prepared by deletion, substitution or addition of one or more aminoacids in SEQ ID No.: 1. As a result, the peptides consisting of SEQ IDNOS:22, 26 or 27 showed better penetrating activity than TAT (100 μM).On the basis of these penetration data, the peptides of SEQ ID NOS:31-45were synthesized repeatedly and these all peptides showed betterpenetrating activity than TAT in 10 μM. On the basis of above data, thepeptides of SEQ ID NOS:46-54 were prepared as various mutant forms ofSEQ ID NO:1, then measured for cell penetrating activity. As a result,the peptide of SEQ ID NO:49 had excellent activity compared with TAT andthe peptides of SEQ ID NOS:46-54 showed a similar or better activitycompared with TAT and excellent activity compared with TCTP(1-10)(SEQ IDNO:1). Therefore, a peptide having cell membrane penetrating activity ofthe present invention comprises the peptides consisting of SEQ ID NO:22,26, 27, or 31-54.

Length of the peptides, as a common length of cell membrane penetratingprotein domain accepted in this art, may vary within the scope of,preferably 5-15 residues, and more preferably 8-10 residues.

The peptide of the present invention may be prepared by artificialsynthesis or by isolating the sequence of TCTP(1-10)(SEQ ID NO:1),TCTP(1-9)(SEQ ID NO:2), TCTP(1-8)(SEQ ID NO:3) or TCTP(2-10)(SEQ IDNO:4) and modifying these sequences.

Synthesis of the peptides may be prepared by same synthesis methods asdescribed above.

The present invention also provides a transmembrane carrier comprisingthe peptide having cell membrane penetrating activity as an effectivecomponent. The peptide having cell membrane penetrating activityprovides a use as a transmembrane carrier for penetrating targetsubstance across plasma membrane.

In addition, the present invention provides a transmembrane complexconsisting of the peptide having cell membrane penetrating activitycombined with a target substance.

The term ‘target substance’ of the present invention means a moleculethat may be related to a regulation of physiological activity, made apharmacological action or maintained a biological activity inintracellular compartment.

Target substance of the present invention, for example, may comprisenucleic acid including DNA and RNA, chemical compound such as drug,carbohydrate, lipid or glycolipid etc. as non-protein range molecule,and enzyme, regulation factor, growth factor, antibody, cytoskeletalfactor etc. as protein range molecule.

A peptide having cell membrane penetrating activity of the presentinvention may be linked to one or more target substances byphysically/chemically covalent bond or non-covalent bond, or bymediators in incorporated or fused forms.

In detail, if the target substance is a non-protein range molecule, apeptide having cell membrane penetrating activity of the presentinvention may be linked to the target substance by covalent bond, thenthe complex may be exposed to target cell group. In another example, thetarget substances may be non-covalently linked to a peptide having cellmembrane penetrating activity of the present invention. For instance, ifthe target substance is a nucleic acid, it may be incorporated with apeptide having cell membrane penetrating activity of the presentinvention, in forms of lipid based vehicle, then exposed to target cellgroup.

In case that the target substance is a protein, fusion proteinincorporated with a peptide having cell membrane penetrating activity ofthe present invention can be prepared by obtaining cDNA of theprotein(the target substance) through PCR and cloning cDNA usingvectors. If it is impossible, the protein may be fused chemically. Forexample, fusion protein can be prepared by connecting the targetsubstance to linker, then reacting with the peptide having cell membranepenetrating activity to form linkage.

In particular, when the target substance is a protein, the complex maybe penetrated in fauns of fusion protein. In this case, cell penetratingcomplex of the present invention may be prepared as follows.

First, recombinant expression vector is prepared to generate a fusiongene encoding a peptide having cell membrane penetrating activity-targetsubstances conjugate.

Nucleic acids encoding above fusion protein include the nucleic acidsequence encoding a peptide having cell membrane penetrating activityand the nucleic acid sequence encoding a protein as target substance.For example, these nucleic acid sequences may comprise sequencesconsisting of SEQ ID NOS:17-18 or 55-81. Nucleic acid sequences of SEQID NOS:17-18 or 55-81 are as follows.

Nucleic Acid Sequences SEQ ID Classification (Homo sapiens) NO:Nucleic acid for SEQ ID NO: 1 (TCTP 1-10) atgattatctaccgggacctcatcagccac17 Nucleic acid for SEQ ID NO: 2 (TCTP1-9) atgattatctaccgggacctcatcagc18 Nucleic acid for SEQ ID NO: 22 (TCTP-CPP#3)atgattatttttcgcgatctgattagccat 55Nucleic acid for SEQ ID NO: 26 (TCTP-CPP#7)atgattatttatcgcgcgctgattagccataaaaaa 56Nucleic acid for SEQ ID NO: 27 (TCTP-CPP#8)atgattatttatcgcattgcggcgagccataaaaaa 57Nucleic acid for SEQ ID NO: 31 (TCTP-CPP#12)atgattatattcgcattgcggcgagccataaaaaa 58Nucleic acid for SEQ ID NO: 32 (TCTP-CPP#13)atgattatttttcgcgcgctgattagccataaaaaa 59Nucleic acid for SEQ ID NO: 33 (TCTP-CPP#14)atgattatttttcgcgcggcggcgagccataaaaaa 60Nucleic acid for SEQ ID NO: 34 (TCTP-CPP#15)tttattatttttcgcattgcggcgagccataaaaaa 61Nucleic acid for SEQ ID NO: 35 (TCTP-CPP#16)ctgattatttttcgcattgcggcgagccataaaaaa 62Nucleic acid for SEQ ID NO: 36 (TCTP-CPP#17)tggattatttttcgcattgcggcgagccataaaaaa 63Nucleic acid for SEQ ID NO: 37 (TCTP-CPP#18)tggattatttttcgcgcggcggcgagccataaaaaa 64Nucleic acid for SEQ ID NO: 38 (TCTP-CPP#19)tggattatttttcgcgcgctgattagccataaaaaa 65Nucleic acid for SEQ ID NO: 39 (TCTP-CPP#20)atgattatttacgcattgcggcgtatcataaaaaa 66Nucleic acid for SEQ ID NO: 40 (TCTP-CPP#21)tggattatttttcgcattgcggcgtatcataaaaaa 67Nucleic acid for SEQ ID NO: 41 (TCTP-CPP#22)atgattatttttcgcattgcggcgacccataaaaaa 68Nucleic acid for SEQ ID NO: 42 (TCTP-CPP#23)tggattatttttcgcattgcggcgacccataaaaaa 69Nucleic acid for SEQ ID NO: 43 (TCTP-CPP#24)atgattatattaaaattgcggcgagccataaaaaa 70Nucleic acid for SEQ ID NO: 44 (TCTP-CPP#25)tggattatttttaaaattgcggcgagccataaaaaa 71Nucleic acid for SEQ ID NO: 45 (TCTP-CPP#26)atgattatttttgcgattgcggcgagccataaaaaa 72Nucleic acid for SEQ ID NO: 46 (TCTP-CPP#27)ctgattatttttcgcattctgattagccataaaaaa 73Nucleic acid for SEQ ID NO: 47 (TCTP-CPP#28)atgattatttttcgcattctgattagccataaaaaa 74Nucleic acid for SEQ ID NO: 48 (TCTP-CPP#29)ctgattatttttcgcattctgattagccatcgccgc 75Nucleic acid for SEQ ID NO: 49 (TCTP-CPP#30)ctgattatttttcgcattctgattagccatcatcat 76Nucleic acid for SEQ ID NO: 50 (TCTP-CPP#31)ctgattatattcgcattctgattagccataaa 77Nucleic acid for SEQ ID NO: 51 (TCTP-CPP#32)ctgattatttttcgcattctgattagccatcgc 78Nucleic acid for SEQ ID NO: 52 (TCTP-CPP#33)ctgattatttttcgcattctgattagccat 79Nucleic acid for SEQ ID NO: 53 (TCTP-CPP#34)ctgattatttttgcgattgcggcgagccataaaaaa 80Nucleic acid for SEQ ID NO: 54 (TCTP-CPP#35)ctgattatttttgcgattctgattagccataaaaaa 81

Since codons encoding one amino acid are several, nucleic acid sequencesencoding the peptide of the present invention include all nucleic acidsequence encoding the peptide of the present invention besides nucleicacid sequences listed in above table.

Recombinant expression vector of the present invention may includeconventional promoter for expression, termination factor, selectionmarker, reporter gene, tag sequence, restriction enzyme recognitionssite, multi-cloning site and so on.

Transfection methods to host using recombinant expression vector of thepresent invention may be a heat shock or electroporation etc. which isknown in the art.

After fusion proteins are expressed under proper conditions intransfected host cell as above, fusion proteins, which consist of apeptide having cell membrane penetrating activity and a protein astarget substance, may be purified by conventional methods known in theart.

In addition, the present invention provides a transfection kitcomprising the peptide having cell membrane penetrating activity and thetarget substance. Transfection kits are optimized systems to introduceeasily DNA/RNA to intracellular compartment of mammalian cell. There areup to now calcium-phosphate method, methods using lipid complex ordextran complex, but limitations are that efficiency of these methods is1/10⁶-1/10² and depend on cell type. To overcome these limitations,transfection kits using the peptide having cell membrane penetratingactivity, may be utilized.

The transfection kit of the present invention may further comprise abinding factor combining the peptide with the target substance. Thebinding factor means specific DNA/RNA sequences includingtranscriptional factor, virus protein, or whole body or a part ofprotein that are capable to bind to target substance. For example, Gal4is a DNA binding factor. Gal4 is a transcriptional factor widely used ineukaryote, prokaryote and virus. DNA/RNA binding factors may be used byvector expressing PTDs and fusion proteins in vivo and in vitro. Also,incorporation between DNA/RNA binding factors and PTDs may beaccomplished by chemical interaction, physical interaction ornoncovalent interaction.

If fusion complexes between a peptide having cell membrane penetratingactivity of the present invention and DNA/RNA are treated outside thecells, it can be overcome both efficiency and limitation depending onthe cell type. Using both a peptide having cell membrane penetratingactivity of the present invention and DNA/RNA binding factors, it iscapable that DNA/RNA is introduced into cytoplasm and nucleus of variouscells in vivo and in vitro. Particularly, introduction method can beaccomplished by various route including intramuscular, intraperitoneal,intravenous, oral, subcutaneous, intracutaneous, intranasal introductionand inhalation.

In addition, target substance may include one or more biologicalregulation substances selected from a group consisting of protein,lipid, carbohydrate or chemical and transfection kits of the presentinvention can introduce above target substance into cytoplasm andnucleus of various cells in vivo and in vitro. Fusion between PTD andtarget substance can be accomplished by chemical, physical covalentinteraction or noncovalent interaction.

Transfection kit of the present invention provides new technology aboutgene therapy and DNA/RNA vaccine according to the methods of the presentinvention and can express transiently or permanently and be used inclinical applications such as gene therapy and DNA/RNA vaccine as wellas basic research.

Also, the present invention provides a use of the peptide having cellmembrane penetrating activity for the manufacture of a transmembranecomplex and a method for preparing transmembrane complexes by combiningtarget substance with the peptide having cell membrane penetratingactivity.

In addition, the present invention provides a use of the transmembranecomplex consisting of the peptide having cell membrane penetratingactivity combined with a target substance for the manufacture of amedicament and a method for manufacturing a medicament which comprisesmixing the transmembrane complex consisting of the peptide having cellmembrane penetrating activity combined with a target substance, with apharmaceutically acceptable carrier. The pharmaceutically acceptablecarrier is well known to a skilled artisan, and the skilled artisan canselect and use the pharmaceutically acceptable carrier which is properfor introduction to a living body.

Further, the present invention provides a method for delivering a targetsubstance into cell interior which comprises administrating to a subjectwith a transmembrane complex consisting of the peptide having cellmembrane penetrating activity combined with a target substance to inducetransduction of the transmembrane complex into cell interior.

If the target substance is non-protein range molecule, it may becovalently attached to the peptide having cell membrane penetratingactivity of the present invention, and the complex may be exposed totarget cell group. In another example, the target substance may benon-covalently attached to the peptide having cell membrane penetratingactivity of the present invention, for example, if the target substanceis a nucleic acid, the complex may be exposed to target cell group informs of lipid based vehicle incorporated with the peptide having cellmembrane penetrating activity of the present invention.

The ‘subject’ may be mammal including human. The transmembrane complexcan be administrated by various route including intramuscular,intraperitoneal, intravenous, oral, subcutaneous, intracutaneous,mucosal administration and inhalation.

Dose of the transmembrane complex consisting of the peptide having cellmembrane penetrating activity combined with a target substance, isvariable according to a therapeutically effective amount of the targetsubstance and penetrating activity of the peptide, and so it is notlimited to a specific dose. Only, for example, if the target substanceis a nucleic acid, the dose of target substance may be 10˜1000 μg/kg andthe dose of the peptide of the present invention may be 0.1 mg-10 mg/kg.

In addition, the present invention provides a method for treatingrelated diseases by administrating to a subject with the transmembranecomplex consisting of the peptide having cell membrane penetratingactivity combined with a target substance thereby introducing the targetsubstance into a cell.

The kind of the disease desired to treatment may be varied depending onthe target substance intended to administrate into cell interior.

The ‘subject’ may be mammal including human. The transmembrane complexcan be administrated by various route including intramuscular,intraperitoneal, intravenous, oral, subcutaneous, intracutaneous,mucosal administration and inhalation.

Also, the present invention provides a nucleic acid sequence encodingthe peptide having cell membrane penetrating activity. For example, thepresent invention provides a nucleic acid encoding the peptide havingcell membrane penetrating activity, consisting of an amino acidsequences selected from SEQ ID NOS:1, 2, 22, 26, 27 or 31-54.

The nucleic acid may be DNA or RNA of single chain or double chain andbe prepared by synthesizing artificially or isolating fromorganism-derived TCTP genes. For example, the nucleic acids encoding thepeptides consisting of SEQ ID NOS:1, 2, 22, 26, 27 or 31-54, representthe nucleic acid sequences of SEQ ID NOS:17-18, or 55-81, respectively.

Since codons encoding one amino acid are several, nucleic acid sequencesencoding the peptide of the present invention include all nucleic acidsequences encoding the peptide of the present invention, and are notlimited to the nucleic acid sequences listed in above table. Forexample, sequence encoding alanine in amino acid sequence may be gca,gcc, gcg or get.

The peptide of the present invention having cell membrane penetratingactivity has a prominent effect in delivery as compared with TAT-derivedpeptide. Thus, the peptide having cell membrane penetrating activity ofthe present invention, the transmembrane complex consisting of thepeptide combined with a target substance, and the method for deliveringa target substance into a cell using the transmembrane complex hasapplications on intracellular delivery in various research fields aswell as on therapeutics of specific diseases where targeting of drugs isrequired at high efficiency. Accordingly, the peptide having cellmembrane penetrating activity of the present invention, thetransmembrane complex consisting of the peptide combined with a targetsubstance, and the method for delivering a target substance into a cellusing the transmembrane complex is very useful as drug delivery systems.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

ADVANTAGEOUS EFFECTS

The peptide having cell membrane penetrating activity of the presentinvention has a prominent penetrating efficiency as compared with theactivities of prior TAT-derived peptides and so the peptide hasapplications in intracellular delivery in various research fields aswell as in therapeutics of specific diseases where targeting of drugs isrequired in high efficiency. Accordingly, the peptide having cellmembrane penetrating activity of the present invention, thetransmembrane complex consisting of the peptide combined with a targetsubstance, and the method for delivering a target substance into a cellusing the transmembrane complex are very useful as drug deliverysystems.

DESCRIPTION OF DRAWINGS

FIG. 1A and FIG. 1C are schematic diagrams showing various deletionforms of TCTP of the present invention, and FIG. 1B and FIG. 1D are thewestern blot analysis results for cellular uptake of the variousdeletion forms of TCTP of FIG. 1A and FIG. 1C in BEAS-2B cell line.

FIG. 2 shows a dose dependent cellular uptake after 15 minutes oftreatment of TCTP-derived peptides.

FIG. 3 shows cellular uptake after 2 hours of treatment of TCTP-derivedpeptides at various concentrations in HeLa cell line.

FIG. 4 shows fluorescence microscope images representing cellular uptakeafter 2 hours of treatment of the TCTP-derived peptides at variousconcentrations in HeLa cell line.

FIG. 5 shows cellular uptakes after 2 hours of treatment of substituentsof TCTP-derived peptide at various concentrations at the sensitivity of75.

FIG. 6 shows same result of FIG. 5 at the sensitivity of 100.

FIG. 7 shows mean fluorescence intensity showing a cellular uptake ofmutant peptides of TCTP-derived peptides(#1-11) treated for 2 hours atvarious concentrations using FACS.

FIG. 8 shows mean fluorescence intensity showing a cellular uptake ofmutant peptides of TCTP-derived peptides(#12-26) treated for 2 hours atvarious concentrations using FACS.

FIG. 9 shows mean fluorescence intensity showing a cellular uptake ofmutant peptides of TCTP-derived peptides(#27-35) treated for 2 hours atvarious concentrations using FACS.

FIG. 10A shows cytotoxicity of mutant peptides of TCTP-derived peptides(#3, #7, #8) treated for 24 hours at various concentrations.

FIG. 10B shows cytotoxicity of mutant peptides of TCTP-derived peptides(#3, #7, #8) treated for 48 hours at various concentrations.

FIG. 11A shows cytotoxicity of mutant peptides of TCTP-derived peptides(#12-26) treated for 24 hours at various concentrations.

FIG. 11B shows cytotoxicity of mutant peptides of TCTP-derived peptides(#12-26) treated for 48 hours at various concentrations.

FIG. 12A shows cytotoxicity of mutant peptides of TCTP-derived peptides(#27-35) treated for 24 hours at various concentrations.

FIG. 12B shows cytotoxicity of mutant peptides of TCTP-derived peptides(#27-35) treated for 48 hours at various concentrations.

MODE FOR INVENTION EXAMPLE 1 Mapping of PTD Using Various Deletion Formsof TCTP

In order to confirm the region of the TCTP acting as PTD, variousdeletion constructs were prepared and then used in the experiment asfollows.

1) Isolation and Purification of Deletion Forms of TCTP

To overexpress each of those deletion forms of TCTP (FIGS. 1 A and 1C),pRSET vector that is capable of tagging 6 histidine was employed.Subcloning with DNA sequences corresponding to each deletion forms ofTCTP was performed in the multicloning site of the vector. Then, therecombinant expression vector was introduced into E. coliBL21(DE3)(Novagen) or BL21(DE3)pLysS (Novagen). The expression of thedeletion forms of TCTP was induced by IPTG (isopropylβ-D-thiogalactoside) for 3 hours, then the protein was isolated andpurified by using Ni column which binds to polyhistidine.

2) Cell Culture and Treatment with the Protein

BEAS-2B cell was treated with the deletion form of TCTP at theconcentration of 15 μg/ml for 1 hour or 24 hours. Then, supernatants andcell lysates were obtained and western blotted with anti-TCTP antibodies(FIG. 1B). As shown in FIG. 1B, full length TCTP existed in cellsupernatants after incubation for 1 hour (Lane 1) but this proteindisappeared 24 hours later (Lane 6). Also, in cell supernatantcontaining Del-C112HRF lacking C-terminus, the protein disappeared 24hours later (Lane 9). On the other hand, remaining deletion forms ofTCTP lacking N-terminus, Del-N11, N35 and N39C110HRF were still existingin cell supernatant 24 hours later (Lane 7, 8, 10).

Therefore, it could be known that PDT of TCTP exists in N-terminusParticularly, since Del-N11HRF was still existed in cell supernatant 24hours later (Lane 7), it seems that TCTP 1-10 plays a role as PDT.

In addition, it was examined whether TCTP proteins of the presentinvention could be transferred to the cellular interior for a shorttime, 5 minutes or 30 minutes. The experiment was performed by samemethod as the above (FIG. 1D).

As shown in FIG. 1D, Del-C38HRF holding N-terminus of HRF disappearedafter 30 minutes (Lane 4) in the supernatant while these proteins werefound after 5 minutes (Lane 1) and 30 minutes (Lane 4) in cell lysates.

Thus, N-terminus containing TCTP proteins of present invention can betransferred into cell interior for a short time, only several minutes toseveral tens minutes.

EXAMPLE 2 Confirmation of Cell Penetrating Efficiency of the Peptide ofthe Present Invention

As shown in Example 1, in order to confirm that the N-terminus of TCTPcan function as a PTD, the peptides consisting of N-terminus of TCTPwere constructed and examined for cell penetrating efficiency.

1) Synthesis of Various Peptides Corresponding N-terminus Amino Acid ofTCTP

TCTP-derived peptides and control peptide, TAT 48-57 were synthesized asfollow.

Sequence SEQ Classification of amino acid ID NO: Residues of TCTP(1-10)MIIYRDLISH  1 Residues of TCTP(1-9) MIIYRDLIS  2 Residues of TCTP(1-8)MIIYRDLI  3 Residues of TCTP(2-10) IIYRDLISH  4 Residues of TCTP(1-7)MIIYRDL  5 Residues of TCTP(1-6) MIIYRD  6 Residues of TCTP(3-10)IYRDLISH  7 Control TAT(48-57) GRKKRRQRRR 19

The N-terminus of each peptide was labeled with fluorescence dye,rhodamine and the C-terminus was protected. Peptide purity (>95%) wasdetermined by HPLC. Synthesis of the peptides was requested to PEPTRON,Inc.

Negative control was a fluorescence dye, rhodamine (Molecular Probe)used to label in all peptides.

2) Cell Culture and Incubation of Peptides

HeLa cell line (ATCC) was propagated in DMEM (GIBCO) supplemented with10% FBS (GIBCO) and 100 units/mL penicillin-streptomycin. Cells weregrown in a 5% CO₂ incubator at 37° C.

HeLa cells were cultured in a 48-well plate until they were 70˜80% grownup before a day of the experiment. The cells were washed with DMEM at37° C. twice, and the TCTP-derived peptides synthesized in Example 2-1were treated to the culture medium in a dose dependent manner (0, 1, 5,10, 50, 100 μM), then the cells were incubated for 15 minutes or 2 hoursin a CO₂ incubator at 37° C.

After the incubation, the cells were washed in cool PBS three times andimmediately measured by a microplate fluorescence reader (BIO-TEKinstruments, Inc., Vermont, USA) at emission 530 nm and excitation 590nm for a measurement of rhodamine of intracellular uptake marker. Thesensitivity of reader was set at 100 as a basic mode, but was lowered to75 if the fluorescent signals were too strong. All experiments wereconducted in triplet repeats for reproducibility (FIG. 2 and FIG. 3).

As shown in FIG. 2 and FIG. 3, TAT, control peptide was transduced intocell in a dose and time-dependent manner as previously known.

TCTP (1-10), (1-9), (1-8) peptides of the present invention weretranslocated not in 1-10 μM but in 50-100 μM at 15 minutes (FIG. 2) or 2hours (FIG. 3). In 50-100 μM, intracellular translocation was observedto be very high and could not detect due to a strong fluorescenceparticularly after 2 hours treatment and thus the sensitivity of readerwas lowered to 75.

In FIG. 3, judging from the fact that there was no difference ontranslocation efficiency between 2 hour treatment at concentration 50 μMand that at 100 μM of TCTP(1-10) peptide, it seemed that TCTP(1-10)peptide was saturated at 50 μM. On the other side, TAT (48-57) peptidewas saturated at 1 μM or more.

TCTP (2-10) peptide was not translocated at a concentrarion of 1 μM to10 μM, but was more efficiently translocated at 100 μM after 15 minutestreatment of this peptide. After 2 hours, this peptide has similar cellmembrane penetrating activity to control peptide, TAT(48-57), and wasmore efficiently translocated at 100 μM than control peptide.

So, it could be confirmed that TCTP (1-10), (1-9), (1-8) and (2-10)peptides having cell membrane penetrating activity of the presentinvention had superior ability than well-known PTD, TAT in theirtranslocation efficiency.

For TCTP-derived peptide, it had been shown a sudden increase intranslocation ability at the high concentration and these results mightbe caused by a difference in translocation mechanisms.

Consequently, it could be confirmed that TCTP (1-10), (1-9), (1-8) and(2-10) peptides having cell membrane penetrating activity of the presentinvention had superior ability than well-known PTD, TAT in theirtranslocation efficiency. From among these peptides, translocationefficiency was superior in the order of TCTP (1-10), (1-9), (1-8) and(2-10) peptides, and existence of methionine (1^(st) amino acid residue)of TCTP N-terminus was important.

EXAMPLE 3 Identification of Intracellular Translocation of TCTP-DerivedPeptide by Fluorescence Microscope

The intracellular translocation of the peptide was identified byfluorescence microscope. HeLa cells were treated with TCTP (1-9)(SEQ IDNO:2) at a concentration of 10 μM and 100 μM by the same method ofExample 2-2. A point of difference was that HeLa cells were seeded in a12 well-plate covered a glass since the plastic plate had a property offluorescence interference. After washing, cells on cover glass attachedslide glass were observed (FIG. 4).

As shown in FIG. 4, the peptide of the present invention was weaklytranslocated at a low concentration of 10 μM and strongly at a highconcentration of 100 μM. It was found that the peptides were distributedwidely in the cytoplasm and nucleus of the cell.

EXAMPLE 4 Identification of Intracellular Translocation of PeptideSubstituents

In order to confirm that substituent forms of the present peptide canfunction as a PTD, substituents of the peptide were constructed andexamined for cell penetrating efficiency.

1) Construction of Peptide Substituents

Serial substituents of TCTP(1-9)(SEQ ID NO:2) with alanine weresynthesized as follows.

Sequence SEQ Classification of amino acid ID NO: TCTP(1-9)M1A AIIYRDLIS 8 TCTP(1-9)I2A MAIYRDLIS  9 TCTP(1-9)I3A MIAYRDLIS 10 TCTP(1-9)Y4AMIIARDLIS 11 TCTP(1-9)R5A MIIYADLIS 12 TCTP(1-9)D6A MIIYRALIS 13TCTP(1-9)L7A MIIYRDAIS 14 TCTP(1-9)I8A MIIYRDLAS 15 TCTP(1-9)S9AMIIYRDLIA 16

N-terminus of each peptide was labeled with fluorescence dye, rhodamineand C-terminus was protected. Peptide purity (>95%) was determined byHPLC. Synthesis of peptides of present invention was requested toPEPTRON, Inc.

2) Cell Culture and Incubation of Peptides

HeLa cell line was propagated in DMEM supplemented with 10% FBS and 100units/mL penicillin-streptomycin. Cells were grown in a 5% CO₂ incubatorat 37° C.

HeLa cells were cultured in a 48-well plate until they were 70˜80% grownup before a day of the experiment. The cells were washed with DMEM at37° C. twice, and the TCTP-derived peptides synthesized in Example 4-1were treated to the culture medium in a dose dependent manner (0, 1, 10,100 μM), then the cells were incubated for 15 minutes or 2 hours in aCO₂ incubator at 37° C.

After the incubation, the cells were washed in cool PBS three times andimmediately measured by a microplate fluorescence reader at emission 530nm and excitation 590 nm for a measurement of rhodamine of intracellularuptake marker. The sensitivity of reader was set at 100 as a basic, butwas lowered to 75 if fluorescent signals were strong. All experimentswere conducted in triplet repeats for reproducibility (FIG. 5 and FIG.6).

As shown in FIG. 5, when fluorescence intensity of TCTP (1-9) at 100 μMwas set to be 100%, the alanine substituents showing the largest declinein uptake were alanine substituents for amino acid residue 1,2,3,4 (eachM, I, I, Y) of TCTP(1-9)(each SEQ ID NOS:8, 9, 10, 11), down by 80-90percent.

On the other hand, alanine substituents for amino acid residue 5, 6, 7,8, 9 (each R, D, L, I, S) of TCTP(1-9)(each SEQ ID NOS:12, 13, 14, 15,16) were declined in uptake, down by about 50 percent but we judged thatthese peptides were still maintained in translocation activity. Thus, itwas known that four amino acids (M, I, I, Y) of the N-terminus of TCTPwere necessary in cell penetrating activity.

Meanwhile, when the sensitivity of KC4 plate reader was set down to 75,we could not analyze the result of cell penetrating activity atrelatively low concentration of 1 or 10 μM, so sensitivity of reader wasfixed at 100 (FIG. 6). At this time, because fluorescence intensity at100 μM was very strong, we could not express in a same graph.

As shown in FIG. 6, when fluorescence intensity of TCTP (1-9) at 10 μMwas set to be 1, alanine substituent for amino acid residue 6th,aspartic acid of TCTP(1-9) (SEQ ID NO:13) had 2.5 times higherpenetrating activity than natural peptide, TCTP(1-9). Aspartic acid is aamino acid with negative charge and only residue having negative chargeof TCTP(1-9). Thus it was considered that amino acid with negativecharge decreased the activity of cell penetration of TCTP.

Natural peptides of TCTP(1-10), (1-9), (1-8), (2-10) were efficientlytranslocated at a high concentration, while these peptides had lowerefficiency than control peptide, TAT at a relatively low concentrationof 1 μM and 10 μM (EXAMPLE 2). However, from the above results it wasshown that analogues of deletion, addition or substitution of 6thresidue had a excellent penetrating activity at a low concentration.

From all of the above results, four amino acids(M, I, I, Y) onN-terminus of TCTP played a necessary role in cell penetrating activityand particularly alanine substituent for 6th residue, aspartic acidincreased suddenly cell penetrating activity at a low concentration (10μM). At this time, we assumed that which penetrating activity wasincreased at a low concentration but decreased at a high concentrationwas due to low solubility of alanine substituent with hydrophobicproperty.

EXAMPLE 5 Cell Penetrating Activity of Mutant Peptides

As shown in EXAMPLE 4, it was confirmed that substituent peptides of thepresent invention had a cell membrane penetrating activity. So toidentify which mutant forms of the present peptides have penetratingactivity, we examined translocation efficiency of mutant peptides.

1) Construction of Mutant Peptides

From the results of EXAMPLE 4, various mutant peptides were constructedwith the frame of TCTP (1-10)(SEQ ID NO:1).

Sequence SEQ Classification of amino acid ID NO: TCTP-CPP#1 MIIYRDLISKK20 TCTP-CPP#2 MIIYRDKKSH 21 TCTP-CPP#3 MIIFRDLISH 22 TCTP-CPP#4MIISRDLISH 23 TCTP-CPP#5 QIISRDLISH 24 TCTP-CPP#6 CIISRDLISH 25TCTP-CPP#7 MIIYRALISHKK 26 TCTP-CPP#8 MIIYRIAASHKK 27 TCTP-CPP#9MIIRRDLISE 28 TCTP-CPP#10 MIIYRAEISH 29 TCTP-CPP#11 MIIYARRAEE 30TCTP-CPP#12 MIIFRIAASHKK 31 TCTP-CPP#13 MIIFRALISHKK 32 TCTP-CPP#14MIIFRAAASHKK 33 TCTP-CPP#15 FIIFRIAASHKK 34 TCTP-CPP#16 LIIFRIAASHKK 35TCTP-CPP#17 WIIFRIAASHKK 36 TCTP-CPP#18 WIIFRAAASHKK 37 TCTP-CPP#19WIIFRALISHKK 38 TCTP-CPP#20 MIIFRIAAYHKK 39 TCTP-CPP#21 WIIFRIAAYHKK 40TCTP-CPP#22 MIIFRIAATHKK 41 TCTP-CPP#23 WIIFRIAATHKK 42 TCTP-CPP#24MIIFKIAASHKK 43 TCTP-CPP#25 WIIFKTAASHKK 44 TCTP-CPP#26 MIIFAIAASHKK 45TCTP-CPP#27 LIIFRILISHKK 46 TCTP-CPP#28 MIIFRILISHKK 47 TCTP-CPP#29LIIFRILISHRR 48 TCTP-CPP#30 LIIFRILISHHH 49 TCTP-CPP#31 LIIRILISHK 50TCTP-CPP#32 LIIFRILISHR 51 TCTP-CPP#33 LIIFRILISH 52 TCTP-CPP#34LIIFAIAASHKK 53 TCTP-CPP#35 LIIFAILISHKK 54

N-terminus of each peptide was labeled with fluorescence dye, FITC andC-terminus was protected. Peptide purity (>95%) was determined by HPLC.Synthesis of the peptides of the present invention was requested toPEPTRON, Inc.

2) Cell Culture and Incubation of Peptides

HeLa cell line was propagated in DMEM supplemented with 10% FBS and 100units/mL penicillin-streptomycin. Cells were grown in a 5% CO₂ incubatorat 37° C.

HeLa cells were cultured in a 6-well plate until they were 70˜80% grownup before a day of the experiment. The cells were washed with DMEM at37° C. twice, and the TCTP-derived peptides synthesized in Example 5-1were treated to the culture medium in a dose dependent manner (0, 1, 10,100 μM), then the cells were incubated for 2 hours in a CO₂ incubator at37° C.

After the incubation, the cells were washed in cool PBS two times andtreated with 1 mg/ml trypsin for 15 min at 37° C. to digest peptidesattached on cell membrane and washed in PBS twice again. Then, the cellswere analyzed by FACS at emission 510 nm and excitation 530 nm for ameasurement of FITC of intracellular uptake marker (FIGS. 7, 8 and 9).Intracellular translocation efficiency of mutant peptides, TCTP-CPP#1-35(SEQ ID NOS:20-54) was compared to wild type(WT), TCTP(1-10)(SEQ IDNO:1) and control peptide, TAT(48-57).

3) Relationship Between Peptide Variants and Cell Penetrating Activity

When mutant peptides were designed, each position of the residues can besubstituted with all 20 amino acids like alanine substitution, but thisis inefficient to search the best effective mutant out of all peptidesbecause charge and isoelectric point of whole peptide after change ofother neighboring position of amino acid also have to be considered.Thus we tried new modification on the basis of the results deduced afterprimary changes then we designed new variant peptides to verify the roleof crucial amino acid. New mutant peptides and sequences were arrangedin the table at EXAMPLE 5-1. We intended to explain the mutated positioneasily by giving a number from I to X (from N-terminus) to each tenamino acid of wild type(WT) (SEQ ID NO:1). To increase the solubilityand binding efficiency of WT to cell membrane(in the same reason of useof polyarginine and polylysine), we did the lysine substitution at theposition of WT-X and simultaneous addition of lysine at the sameposition (SEQ ID NO:20), two lysine substitutions at the position ofWT-VILVIII (SEQ ID NO:21) and two lysine additions to WT (SEQ IDNO:26)(SEQ ID NO:27). Only SEQ ID NO:26 and SEQ ID NO:27 of thesevariants increased cell penetrating activity. According to resultscomparing and analyzing mean fluorescence intensity (MFI) when MFI of WTat the concentration of 10 μM was set to 1, TAT, SEQ ID NO:26 and SEQ IDNO:27 were 6.1 times, 6.04 times and 1.73 times higher than WT at theconcentration of 10 μM, respectively, and TAT, SEQ ID NO:26 and SEQ IDNO:27 were 94.75 times, 144.6 times and 342.9 times higher than WT atthe concentration of 100 _(j)iM in cell penetrating activity,respectively. Therefore variant peptides of all 12 amino acids addingtwo lysines at C-terminus of WT was maintained in next designed variantpeptides(from SEQ ID NO:31) and substitution with other basic aminoacids than lysine and change of number of basic amino acids weretested(SEQ ID NOS:48-52). As a result, additions of 1 or 2 basic aminoacid at the C-terminus showed higher efficiency than WT.

To analyze the role of sulfur of methionine in the position of WT-I, wesubstituted methionine(M) with glutamine(Q) or cysteine(C)(comparisonwith SEQ ID NO:23 and SEQ ID NOS:24-25). As a result, sulfur didn't playa crucial role and so to test the role of hydrophobicity of methionine,methionine was substituted by phenylalanine(F), leucine(L) ortryptophan(W) (comparison with SEQ ID NO:31 and SEQ ID NOS:34-36,comparison with SEQ ID NO:32 and SEQ ID NO:38, comparison with SEQ IDNO:33 and SEQ ID NO:37, comparison with SEQ ID NO:39 and SEQ ID NO:40,comparison with SEQ ID NO:41 and SEQ ID NO:42, comparison with SEQ IDNO:43 and SEQ ID NO:44, comparison with SEQ ID NO:46 and SEQ ID NO:47).Consequently, cell penetrating activities of SEQ ID NOS:37, 38 and 39were lower than SEQ ID NO:34 at the concentration of 100 μM but were52.0 times, 55.6 times and 25.0 times higher than WT in theconcentration of 10 μM, respectively, and so these peptides had anexcellent translocation efficiency in comparison with SEQ ID NO:31 (29times higher than WT). As results of SEQ ID NO:38 in comparison with SEQID NO:32 and SEQ ID NO:37 in comparison with SEQ ID NO:33, substitutionfor tryptophan did not increase translocation efficiency. This resultmight be related to cytotoxicity of tryptophan substituents at theconcentration of 100 μM (FIGS. 11A and 11B). In comparison between SEQID NOS:39 and 40, SEQ ID NOS:41 and 42, SEQ ID NOS:43 and 44,substitution for tryptophan instead of methionine did not induce theimportant changes in the aspect of efficiency and cytotoxicity.Substitution for phenylalanine (SEQ ID NO:34) or leucine (SEQ ID NO:35)brought about the increased result of translocation efficiency at theconcentration of 10 μM and a decreased result at 100 μM, compared to SEQID NO:31. Leucine substituents in SEQ ID NOS:31, 34, 35 and 36 causedthe most increased result at 10 μM and the little decreased result at100 μM. Cytotoxicity of SEQ ID NO:35 was weaker than SEQ ID NO:31 at 100μM. In SEQ ID NO:46 (3.75 times higher than MFI of WT 10 μM) and SEQ IDNO:47 (7.04 times higher than MFI of WT 10 μM), substitution for leucinecaused the decreased penetrating activity but toxicity of SEQ ID NO:46was weaker than that of SEQ ID NO:47. Considering problems of methioninewith cytotoxicity and reduction instability, we judged it was mostappropriate that methionine was substituted by leucine and so introducedleucine in peptide variants after this experiment (From SEQ ID NO:48).

To test the role of tyrosine(Y) at the position of WT-IV, bysubstituting tyrosine with phenylalanine(F) having no hydroxyl group butisostericity like a tyrosine or serine(S) having hydroxyl group like atyrosine, we tested the importance of hydrophobicity and the action ofhydroxyl group and so on in this position. SEQ ID NOS:22 and 25 were19.63 times and 0.91 times higher than WT at 10 μM and 216.75 times and1.81 times higher at 100 μM, respectively. From this result, it wasknown that increase of hydrophobicity enhanced cell penetrating activityin this position, and so after this experiment we introducedphenylalanine in the position of WT-IV of peptide variants (From SEQ IDNO:31).

We compared substituents for basic amino acid by substitutingarginine(R) with lysine(comparison between SEQ ID NOS:31 and 43, andbetween SEQ ID NOS:36 and 44) or alanine (comparison between SEQ IDNOS:31 and 45 and between SEQ ID NOS:35 and 53) in the position of WT-V.As a result, translocation efficiency of SEQ ID NO:31 (26.77 timesincrease in comparison with WT) was lower than SEQ ID NO:43 (12.1 timesincrease) and efficiency of SEQ ID NO:36 (18.4 times increase incomparison with WT) was lower than SEQ ID NO:44 (15.04 times increase)at 10 μM. Translocation efficiency of SEQ ID NO:45 (11.47 times increasein comparison with WT) and SEQ ID NO:53 (8.24 times increase incomparison with WT) was lower than SEQ ID NOS:31 and 35 (29.53 timesincrease) at 10 μM. From these results, we thought that maintenance ofthe arginine at position of WT-V had advantages.

Aspartic acid at the position of WT-VI, because SEQ ID NO:13 had a goodefficiency at the low concentration (EXAMPLE 4), was substituted byalanine or isoleucine to increase hydrophobicity. In comparison betweenSEQ ID NO:31 (WT-VI:I) and SEQ ID NO:33 (WT-VI:A), translocationefficiencies of both was similarly increased at 100 μM but sinceincreased penetrating activity of SEQ ID NO:31 (29 times increase incomparison with WT) was far better than SEQ ID NO:33 (3.2 times increasein comparison with WT) at 10 μM, isoleucine substitution was moreeffective than alanine substitution. From these results, after thisexperiment, isoleucine was introduced at the position of WT-VI ofpeptide variant (from SEQ ID NOS:31, 34-36, 39).

When leucine and isoleucine at the position of WT-VII and VIII weresubstituted by alanine respectively (SEQ ID NOS:14 and 15), cellpenetrating activity was decreased and when both were substituted bybasic amino acids, this activity was decreased twice (in the comparisonbetween SEQ ID NOS:1 and 21) and when only leucine at the position ofWT-VII were substituted by glutamic acid(E) having negative charge withstrong hydrophilicity, this activity was decreased to same degree withalanine substituent (in the comparison between SEQ ID NOS:1 and 29) andthus it was concluded that most effective amino acids in both positionswere leucine and isoleucine.

Serine at the position of WT-IX, when SEQ ID NO:39 (WT-IX:Y) and SEQ IDNO:41 (WT-IX:T) substituted by each tyrosine and threonine only at thisposition were compare with SEQ ID NO:31 (WT-IX:S) in cell penetratingactivity, should be maintained for the best effect. Meanwhile in allcase of substitution for tryptophan instead of methionine at theposition of WT-I, efficiency of SEQ ID NO:36 (WT-IX:S) was stronger thanSEQ ID NO:40 (WT-IX:Y) and SEQ ID NO:42 (WT-IX:T) only at 10 μM.

It was effective to maintain histidine(H) at the position of WT-X. Incomparison cell penetrating activity between SEQ ID NOS:1 and 2(deletion of histidine from SEQ ID NO:1), SEQ ID NO:1 was more effectivethan SEQ ID NO:2 at the concentration of 50 μM (See FIGS. 2 & 3), andwhen histidine was substituted by glutamic acid (comparing SEQ ID NO:1with SEQ ID NOS:28 and 30, See FIGS. 7, 8 & 9), SEQ ID NO:28 and SEQ IDNO:30 were similar with WT at 10 μM and decreased 4-5 times at highconcentration.

EXAMPLE 6 Identification of Cytotoxicity of Mutant Peptides

To confirm whether cell penetrating activity of the peptides of thepresent invention was due to membrane weakness as a result ofcytotoxicity, we measured cytotoxicity as follows. HeLa cells werecultured in a 96-well plate until they were 70% grown up before a day ofthe experiment. Control TAT 48-57 and the mutant peptides atconcentrations of 0, 1, 10, 100 μM were treated to DMEM supplementedwith 10% FBS for 24 and 48 hours. After 2 hours in addition of 10 μl ofCCK-8 to each well, absorbance at 450 nm was measured by KC4 platereader (FIGS. 10A, 10B, 11A, 11B, 12A and 12B). As a result of toxicityat 100 μM for 24 hours, cytotoxicity of SEQ ID NO:1, TCTP(1-10) wasabout 14% compared with control, and cytotoxicities of the otherpeptides, TCTP-CPP#3, 7 and 8 were insignificant considering standarddeviation. When treated for 48 hours, all peptides had no cytotoxicityat 1 μM and 10 μM while cytotoxicities of TAT, TCTP(1-10), TCTP-CPP#3, 7and 8 were about 53.8, 28.3, 46.2, 8.2 and 25.6%, respectively. All ofTCTP-CPP#12-26 had no cytotoxicity at 1 μM and 10 μM, but hadcytotoxicity beside only TCTP-CPP#26 at 100 μM. Also, all ofTCTP-CPP#27-35 had no cytotoxicity at 1 μM and 10 μM but hadcytotoxicity at 100 μM.

1. A method for delivering a target substance into a cell, comprisingadministering a transmembrane complex to a subject, wherein the complexcomprises: a) a protein transduction domain (PTD) peptide that consistsof the sequence of amino acids set forth in any of SEQ ID NOS: 39, 41,43, 45, 26 and 27; and b) a target substance that is linked to the PTDpeptide, wherein: the target substance is heterologous to the PTDpeptide; and the PTD peptide is linked to the target substance fordelivery of the target substance into the interior of a cell, wherebythe target substance is delivered into the cell.
 2. The method of claim1, wherein the target substance is selected from among a nucleic acid, adrug, a chemical compound, a carbohydrate, a lipid, a glycolipid, anenzyme, a regulating factor, a growth factor and an antibody.
 3. Themethod of claim 1, wherein the PTD peptide is linked to the targetsubstance via a linker.